The primary objective of this investigation is to study the mechanism of RNA biosynthesis in Escherichia coli and in bacteriophage-infected E. coli. The major emphasis during the current project period will be on the structure, function and mechanism of action of the rho transcription termination protein. The enzymatic properties of the RNA-dependent ATPase activity of rho will be studied. This will include an analysis of the kinetics of the reaction, the effects of inhibitors and the minimum requirements for an RNA to activate the ATPase activity. An attempt will be made to define the role of the ATPase reaction in the termination of RNA synthesis catalyzed by RNA polymerase. An analysis will be made of the ATP-dependent RNA release activity of rho for the RNA in isolated ternary transcription complexes of DNA, nascent RNA and RNA polymerase. The mechanism of rho action in termination will be investigated in detail using specific restriction endonuclease-generated fragments of bacteriophage gamma DNA that code for the synthesis of a single RNA transcript that can be terminated by rho action. Other aspects of the mechanism of rho action will be studied from the interaction between rho and poly(C) and the effects of the presence of ATP and other nucleotides on this interaction. Finally some attempts will be made to determine whether rho has some function in regulating RNA synthesis in bacteria under conditions of physiological stress.